Membrane Proteins have long been recognized as major drug targets, and delineating their structure and function have been strong foci. However, a smaller percentage of stalwart researchers have been dauntless enough to tackle the challenges of extracting, expressing and purifying these formidable proteins. In particular, removing membrane proteins from their hydrophobic environment and keeping them stable in an aqueous state is a significant accomplishment that will be discussed in this meeting both through case studies and through sharing of protocols.

Though yields of membrane protein production only reach into milligrams, this innovative work holds great promise for future research that may subsequently result in new therapies and drugs. Join this indepth exploration of how to obtain functional membrane proteins, and learn more about this important protein class.

Tuesday, January 12

Arrive early and join BuzZ Session Roundtable Discussions, Poster Awards and Short Courses!

Lawrence DeLucas, Ph.D., Center for Biophysical Sciences and Engineering, University of Alabama at Birmingham

This presentation will review major hurdles that must be overcome to produce milligram quantities of homogeneous, stable and biologically relevant eukaryotic membrane proteins. Discussion of various approaches that led to successful outcomes for specific protein classes will be followed by introduction of a novel approach to express, solubilize, and stabilize membrane proteins.

Chemokine receptors play many important physiological roles in the body, but are also implicated in inflammatory diseases, cancer and HIV, making them attractive therapeutic targets. However, like many other membrane proteins, biochemical and structural studies of these GPCRs are hampered by the difficulties of obtaining high levels of pure, functional receptor.

Size-exclusion chromatography (SEC) coupled with “on-line” static laser light scattering (LS), refractive index (RI), and ultraviolet (UV) detection allows determination of the association state of proteins in solution. In the SEC-UV/LS/RI approach, SEC serves solely as a fractionation step while the responses from the three detectors are utilized to calculate the molar mass for the polypeptide portion of the protein. The amount of lipid, or detergent bound to the polypeptide chain can also be estimated from the SEC-UV/LS/RI analysis. SEC-LS/RI/UV does not require any adjustment to the composition of buffer during the assessment of monodispersity of the protein-detergent complex.

10:00 Networking Coffee Break in the Exhibit Hall

G-Protein Coupled Receptors (GPCRs)

10:45 De novo Synthesis of a Functional GPCR Supported in a Nanolipoprotein Disc

Functional characterization of GPCR proteins was accomplished by the use of fluorescent correlation spectroscopy, which can provide information on both qualitative and quantitative assessment of membrane proteins never before obtained using other biophysical techniques. For instance, with little or no purification we can readily measure structure dynamics as well as ligand and protein interactions associated with the NLP-GPCR complex. Our combined approach represents a unique solution to solubility, purification, and characterization problems normally associated with membrane proteins.

Here, I will highlight our studies of expression G-protein coupled receptors (GPCRs), in which we try to understand how the membrane proteins form and insert into the membrane of our heterologous host, S. cerevisiae, as well as characterize their properties once they are isolated in membrane-mimetic environments. Over the last eight years, we have had great success expressing the human adenosine A2a (A2aR) in yeast. Our current focus is on the translocation and topology of these receptors in the ER membrane, which we believe to be the limiting step to folding and proper localization.

The peripheral cannabinoid receptor, CB2, was expressed in Escherichia coli as a fusion with maltose binding protein and several affinity tags. We identified critical factors, namely cholesteryl hemisuccinate and specific cannabinoid ligands, required for stabilization of the receptor in detergent micelles and preservation its structural integrity during solubilization and purification. A procedure for incorporation of the purified receptor into a lipid bilayer was optimized to yield milligram quantities of fully functional CB2 reconstituted into liposomes of a defined composition.

12:15 pm Close of Morning Session

12:30 Lunch on Your Own

ENHANCING EXPRESSION

2:00 Chairperson’s Remarks

Lawrence DeLucas, Ph.D., Center for Biophysical Sciences and Engineering, University of Alabama at Birmingham

2:05 E. Coli Mutants Selected for Improved Protein Expression

James U. Bowie, Ph.D., Professor, University of California, Los Angeles

We have developed a simple genetic selection for improved protein expression. The selection is powerful enough to isolate host mutations that increase expression of membrane targets, including G-protein-coupled receptors. I will describe our progress in obtaining E. coli strains with improved expression capabilities.

2:35 Membrane Protein Expression: Where Quality and Quantity Meet

Jian Payandeh, Ph.D., Department of Pharmacology, University of Washington

Achieving high-level expression of integral membrane proteins remains an important challenge for structural biology. I will describe projects targeting bacterial and eukaryotic membrane proteins that have both succeeded and stalled. I will also highlight general expression and engineering strategies that may be considered, along with examples where the downstream sample quality has ultimately dictated the expression system of choice.

3:05 Protocol Development for the Preparative Scale Cell-Free Production of High Quality Membrane Proteins

By implementing a robotic process for the throughput optimization of target specific expression protocols we demonstrate the cell-free production of more than 150 targets of diverse origin in preparative scale amounts. The quality of selected membrane proteins including large eukaryotic solute carriers, G-protein coupled receptors as well as membrane integrated proteases involved in Alzheimer’s disease has been evaluated and verified by a number of complementary techniques.

Obtaining high yields of soluble and functional recombinant membrane proteins. is a critical challenge which we addressed by the integration of Wheat Germ TNT cell-free expression and HaloTag7 based purification. The Wheat Germ cell-free provides expression of full length functional proteins, while HaloTag7 further enhances expression and provides efficient protein purification through covalent immobilization coupled with proteolytic tag removal. We have expressed and purified a panel of membrane proteins, including GPCR’s, to high yield and purity while maintaining their functionality.

In this presentation, applications of purified membrane proteins in the drug development process will be discussed. I will present a case study of expression, purification, and applications of membrane proteins including the methodology and infrastructure for membrane protein purification.

5:00 Ligand Binding Properties and Stability in Solution after Purification of the Human Hedgehog Receptors Patched (PTC) and Smoothened (SMO) Expressed in Yeast

We expressed the human receptors of the Hedgehog pathway Patched (a 12 TMD putative transporter) and Smoothened (from the GPCR family) in the yeast S. cerevisiae and demonstrated that both receptors are able to bind their ligand in the yeast membrane. We showed that both membrane proteins retained ligand binding properties after purification and we studied their stability in different surfactants.